The Porcine Parvovirus (PPV) causes reproductive failure in swine, resulting in death and foetal mummification, still births and other reproductive failures in pregnant sows. (Joo & Johnson. 1976. Veterinary Bulletin 46, 653-660; Mengeling. 1978. J. Am. Vet. Med. Assoc. 172, 1291-1294). PPV is an autonomous parvovirus containing a single strand DNA molecule of approximately 5000 nucleotides (Mollitor et al. 1984. Virology 137, 241-254). The complete sequence of this genome has been recently described by our group (Ranz et al. 1989. J. Gen. Virol. 70, 2541-2553). Four virus-specific proteins have been described: three capsid proteins (VP1, VP2 and VP3 of Mr values 83000, 64000 and 60000 daltons, respectively) and one non structural protein NS1.
The PPV is related to the Kilham rat virus (KRV) group of autonomous parvoviruses formed by KRV, minute virus of mice (MVM), LuIII, H-1, Feline Panleukopenia virus (FPV), canine parvovirus (CPV) and the mink enteritis virus (MEV). These viruses share several common features with other autonomous parvoviruses:
1. There are two large open reading frames (ORFs). PA1 2. The mRNAs from both ORFs are polyadenylated and 3'-coterminal. PA1 3. The left ORF encodes non-capsid proteins which are necessary for viral DNA replication and the right ORF encodes the major capsid proteins as a nested set. PA1 A. Ranz, J. J. Manclus, E. Diaz Aroca, J. I. Casal. 1989. Porcine Parvovirus: DNA sequence and genome organization. J. Gen. virol. 70, 2541-25463. PA1 J. I. Casal, E. Diaz, A. Ranz, J. J. Manclus. 1990. Construction of an infectious genomic clone of PPV. Effect of the 5' end on DNA replication. Virology 177, 764-767. PA1 Luckow, V. A. & Summers, M. D. 1988. Trends in the development of baculovirus expression vectors. Bio/Technology 6, 47-55. PA1 Vialard et al. 1990. Synthesis of the membrane fusion and hemagglutinin proteins of Measles virus using a novel baculovirus vector containing the .beta.-galactosidase gen. J. Virol. 64, 37-50. PA1 1. The PPV VP2 protein produced in an expression vector of baculoviruses multiplied in a cell culture of a permissive host (this protein hereinafter optionally refered to as "VP2 hereof") or PA1 2. Empty VP2 capsids formed by assembly of the VP2 hereof.
To date, there are several vaccines protecting from porcine parvovirus disease, which are based on conventional inactivation methods of the virus. However, every previous attempt of new vaccines production using recombinant proteins produced in procariotic microorganisms (v.g. E. coli) have failed. In this invention, a new process is described for obtaining a new kind of vaccines based on the immunogenic properties of the major protein VP2, expressed in a baculovirus system multiplied in a cell culture of a permissive host.
For the last years, our laboratory has been studying with great detail the molecular biology of PPV. The findings obtained thus are summarized in two pioneer publications:
These publications are related with the knowledge of the viral DNA sequences encoding the proteins forming the vital capsid. These sequences allowed the identification of the gene that encodes the VP2 of PPV and its manipulation and insertion into the specific vectors to be expressed in the baculovirus system. This system allows a large-scale protein production based upon the replication of recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcMNPV) in insect cell cultures. The state of the art for these vectors is summed up in two scientific papers as follows:
The advantages of VP2 protein synthesis in a baculovirus vector are remarkable over the virus production in cell culture and subsequent purification, in the economic cost of the process and immunogenic antigen output. On the other hand, this invention avoids the sacrifice of animals to stablish primary cell cultures for virus replication, to keep viral reservoires and the usual hazard in virus handling, etc.